1. Field of the Invention
The invention relates to a method of leak testing of hand-held, single-channel or multichannel piston stroke pipettes, preferably of those with air cushions. The subject matter of the invention is also a leak testing apparatus which is suitable for carrying out this method. The subject matter of the invention is finally a system which includes the corresponding leak testing apparatus.
2. Description of Related Art
Piston stroke pipettes are likewise known as hand-held single channel measurement devices (German Patent Application DE 39 03 241 A1) as well as multichannel measurement devices (German Patent Application DE 100 13 511 A1 and corresponding U.S. Pat. No. 6,627,160 B2). In the process of aspiration of a sample liquid, negative pressure is produced in the piston stroke pipette and in the pipette tip which is slipped onto a slip-on section of the pipette. Sample liquid is sucked into the pipette tip by this negative pressure. Piston stroke pipettes, as direct displacers, have no air volume or only a minimum air volume above the liquid level in the pipette tip. They are less suited for biological samples since, there, it is necessary to work largely free of contamination. Therefore, air cushion piston stroke pipettes are more common in which there is a distance between the bottom of the piston and the liquid level such that the liquid level is always only in the interchangeable pipette tip which is generally made as a disposable part, while the piston bottom moves distinctly above the liquid level and thus is protected against contamination.
In piston stroke pipettes and especially in air cushion piston stroke pipettes, the requirement of tightness of all connecting sites of the pipette and the pipette tip is very strict. Due to the differential pressure during suction, air can unintentionally flow into the dead space above the liquid level via a leak. The volume which has been enlarged by the piston stroke then no longer agrees with the actually changed volume.
The sealing sites of piston stroke pipettes are subject to wear during operation. Ageing, chemical attack and other ambient influences have adverse effects, even for static seals. One especially sensitive sealing site, in addition to the dynamic piston seal, is the connection between the pipette tip and pipette on the slip-on section. For hand-held piston stroke pipettes, the process of slipping on the pipette tip takes place manually and essentially free-handed. The surfaces of the slip-on section and pipette tip are highly stressed in doing so. Moreover, new pipette tips are always being used (disposal part) so that the production tolerances of the pipette tips and the surface quality of the sealing surfaces of the pipette tips become noticeable. As a result, after longer use ridges form on the slip-on section of the pipette which can ultimately lead to a leak. Therefore, a slowly rising leakiness is characteristic for piston stroke pipettes.
Not every leak leads to a loss of serviceability of a piston stroke pipette. A very small leak can be tolerated. If the leak has only a very small flow cross section, air flows only very slowly into the dead volume of the pipette and the leak can still be within the framework of the acceptable fault tolerance.
For piston stroke pipettes, it is thus part of normal laboratory operation that the pipettes are subjected to leak testing from time to time. This is regardless of whether they are hand-held piston stroke pipettes or automatic pipetting devices. Even in the latter, there is still a corresponding risk of leaks, all the more considering that observation is limited or not possible at all.
For automatic pipetting devices, in the past, only an optical leak test was performed and such testing was only able to identify coarse leaks. According to this traditional method, the operation of the automatic pipetting device is temporarily stopped after the pipette tip into which the sample liquid has been sucked has been moved upward from the liquid level. An operator visually checks whether sample liquid is dripping from the pipette tip. For this method, a highly experienced operator is required. Moreover, this method can only be used for large leaks and large amounts of liquid. For small volumes of sample liquid, for example, of 10 μl, this method fails.
Therefore, for an automatic pipetting device, an automated method for leak testing has already been proposed (German Patent DE 693 26 773 T2) in which the pressure which is prevailing in the pipetting device, therefore in the dead volume of the pipette and pipette tip, is measured and evaluated in different pipetting states. For this purpose, the pipetting device is itself provided with a pressure sensor which detects the pressure in the dead volume between the liquid level and the piston bottom of the piston pump of the pipette. An electronic control apparatus compares the internal pressure which has been detected by the pressure sensor after taking in of the sample liquid relative to the atmospheric pressure of the ambient atmosphere. In this way, a major leak can be ascertained which makes aspiration of the sample liquid more or less completely impossible. In a second step, the internal pressure with the piston pump stopped after a certain time interval is measured and compared to the internal pressure which prevailed immediately after the end of aspiration. Depending on the pressure rise which has been determined in this method, a leak warning for the automatic pipetting device is delivered or the automatic pipetting device is released for further precision pipetting.
In the above explained known method for leak testing, in an automatic pipetting device, typical volumes of the sample liquid taken in during the leak test are 200 to 500 μl distilled water. The time interval during which pressure in the dead volume of the pipette is measured is typically 30 s compared to the first measurement immediately after stopping the pump after roughly 500 ms. This patent does not disclose anything about pressure conditions.
A method for leak testing in automatic pipetting devices cannot be applied to hand-held piston stroke pipettes, preferably air cushion piston stroke pipettes. To do this, the pipette itself would have to be equipped with a corresponding leak testing apparatus. This cannot be justified in terms of costs and would not be commercially acceptable. For this reason, manual methods for leak testing which can be carried out externally are used for hand-held piston stroke pipettes.
In a known method (Eppendorf, Pipettentester 4708), a bottle which stores a liquid, a filled hose connected to it, and an adapter are used. The adapter is connected to the attached pipette tip. The pipette arranged with the pipette tip up. The liquid level which is settling is marked. After several actuations of the piston of the pipette the position of the liquid level is checked again, the pipette having to be held exactly at the same height. If the liquid level has been established at a lower level, the system of pipette and pipette tip is leaky.
Another similar method is also known in which a hose which leads to a leak testing apparatus is connected to the pipette tip which has been slipped onto the pipette. The leak testing apparatus has a hand-actuated piston pump which produces a negative pressure and an analogously operating manometer which is connected to the piston pump on the intake side as a pressure sensor. The vacuum pump is actuated by hand until a sufficient measurement pressure below atmospheric pressure is established which is shown on the manometer. In this position, it is attempted to fix the vacuum pump by hand so that its pump volume no longer changes. Then, the pipette is repeatedly actuated with the other hand. If afterwards the manometer shows a higher pressure than previously, a leak in the system of pipette and pipette tip must be concluded to exist.
The method for leak testing of hand-held, single-channel or multichannel piston stroke pipettes, preferably of those with air cushions, which is carried out with the above explained device constitutes the point of departure for the teaching of this application.
In addition to the above explained known method, a method is known which works with a special measurement pipette (German Utility Model DE 82 30 186 U1). Here, a measurement pipette is connected to the pipette via an adapter. The sample liquid is taken up directly by the measurement pipette. The taken-up volume can be read directly on its scale. This volume can be compared to the setpoint which has been set on the pipette. In this method, it is disadvantageous that the air cushion, therefore the dead volume, does not agree with the air cushion which is actually present in the pipette in normal operation. The hydrostatic pressure of the taken-up liquid is also greater. Consequently, the method with use of the measurement pipette does not correspond to the actual method in working with the pipette itself.
Studies have shown that different parameters arise in pipetting with different pipettes and pipette tips which influence the results of leak testing in wide limits. The operator must ascertain by experience which parameters must be maintained and to what extent. Such a parameter is the minimum negative pressure below atmospheric pressure which forms in the intake process (see the explanation of German Patent Application DE 39 03 241 A1 above). The viscosity of the sample liquid, the strength of the reset spring which acts on the piston of the piston pump and the flow cross section of the pipette tip influence the negative pressure which is being established. The pipette should be tested at least with this pressure in order to correctly detect the amount of gas flowing in at the leak and in a manner suitable for practice. Another parameter is the pressure rise over a certain time interval. It is dependent on the size of the dead volume in the unit consisting of the pipette and pipette tip during the leak test. Even the initially explained method for leak testing in an automatic pipetting device does not take this quantity into account. Consequently a lowermost boundary value must be stipulated itself for the sake of reliability. The result is that pipettes which are in fact still usable are also serviced.